Abstract Kidney transplantation is the treatment modality of choice for the majority of patients suffering from end stage renal disease, with widely accepted benefits in patient survival and quality of life. Mounting evidence suggests that donor-specific antibodies (DSAs) are a major barrier to improved long-term outcomes following kidney transplantation. Costimulation blockade therapy with CTLA-4Ig has shown promise as a potential therapeutic strategy to control DSAs, showing reductions in the incidence of DSA titers in the post-transplant phase compared to traditional calcenurin targeted immunosuppression. T follicular helper (Tfh) cells, a subset of CD4+ T cells required for optimal antibody production, are reliant on the CD28 costimulatory pathway. CTLA-4Ig inhibits T cell activation by binding the ligands CD80 and CD86 to prevent their engagement with the T cell co-stimulator CD28. Undesirably, this mechanism of action also indiscriminately blocks CD80 and CD86 ligation of the co- inhibitor CTLA-4, depriving T cells of important inhibitory signals and interactions. Our group has demonstrated that use of selective CD28 domain antibody (dAb) that directly antagonizes CD28 affords improved graft survival and suppression of Tfh cell-mediated DSA responses compared to CTLA-4Ig. Interestingly, donor-reactive Tfh cells differentially upregulated CTLA4 expression, suggesting a potential role for CTLA-4 in mediating the superior inhibition observed with the anti-CD28 dAb. Therefore, within this proposal we aim to determine the requirement of coinhibitory signaling through CTLA-4 and PD-L1 in the improved suppression of Tfh-mediated responses following transplantation exhibited by selective CD28 blockade. In vivo experiments will be performed investigating the effect of pharmacological blockade of CTLA-4 and PD-L1 in conjunction with CD28 dAb treatment to observe any changes in the efficacy of graft survival and Tfh cell-mediated allo-immune responses. Further in vivo experiments utilizing the inducible conditional gene knockout Cre-Lox system will then be performed to determine the intrinsic or extrinsic role of CTLA-4 and or PD-L1 in the enhancement of selective CD28 blockade. The proposed research project will serve as a framework for the applicant?s training goals of integrating basic science research in transplantation immunology into a career as an academic scientist whose research interest will focus on developing new strategies of immunosuppression for the induction of graft-specific tolerance. These studies will address the treatment of end stage kidney disease with renal transplantation using a novel immunomodulatory strategy to better suppress T cell dependent and Tfh driven donor-specific antibody responses.